Methods for separating biomolecules, such as nucleic acids and proteins, by size are fundamental to research and diagnostic activities.
Conventionally, separation is carried out by electrophoresis, that is to say by displacement of the molecules under the effect of an electric field. Electrophoresis is conventionally carried out in a matrix, which can be a gel or a polymer solution.
However, conventional gel electrophoresis requires large quantities of material, is of long duration and has a limited resolution for large molecule sizes (for example for DNA molecules of more than 40 or 50 kb). The use of a pulsed field allows the resolution power for molecules of large size to be improved, but it is carried out over an even longer duration (greater than 10 hours).
Electrophoresis carried out in capillary tubes is more rapid and more effective than conventional gel electrophoresis. However, the introduction of matrices into the capillary tubes is problematic. The cross-linking of gels in situ is difficult to reproduce, and the use of polymer solutions of high viscosity requires the application of a very high pressure for filling and emptying the tubes.
It is therefore desirable to have available methods for separating biomolecules by size which do not use separating matrices.
The article entitled “Mechanism for the separation of large molecules based on radial migration in capillary electrophoresis” by Zheng & Yeung in Anal. Chem. 75:3675-3680 (2003) describes a technique for separating DNA in a capillary tube having an inside diameter of 75 μm without a separating matrix, based on the phenomenon of radial migration. This phenomenon consists in displacing the DNA molecules either towards the centre of the capillary or towards its periphery, by joint application of a hydrodynamic flow and an electric field, as a function of the relative direction of the hydrodynamic flow and of the electric field. The authors state that the speed of radial migration depends on the size of the molecules. Accordingly, they propose a separation by size of two molecules of approximately 48 kb and 5 kb by electrophoresis combined with the cyclic application of a hydrodynamic flow of alternating direction. The use of a constant electric field and a constant hydrodynamic flow, on the other hand, does not permit a satisfactory resolution.
The article entitled “Single molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA” by Liu et al. in J. Am. Chem. Soc. 133:6898-6901 (2011) describes a separation of DNA molecules by size in a solution without a matrix, inside a capillary tube having an inside diameter of 2 μm, which is of purely hydrodynamic type.
The article “Free-solution oligonucleotide separation in nanoscale channels” by Pennathur et al. in Anal. Chem. 79:8316-8322 (2007) describes a method of separating DNA molecules of small size (10 to 100 bp) in nanochannels, under the effect of an electric field and without a separating matrix. The authors explain that the migration of the molecules depends especially on the interactions thereof with the electrical double layer, on the steric hindrance and on the presence of end labelling with a fluorescein group (which alters the electrophoretic mobility of the molecules of small size).
The review “Resolving DNA in free solution” by Wang et al. in Trends in Analytical Chemistry, 35:122-134 (2012) describes a number of approaches for separating DNA by size without a matrix. In addition to the technique of Zheng & Yeung mentioned above, the review describes techniques of electrophoresis with end labelling of the molecules (which requires a charge to be attached to the DNA molecules at their ends), of entropic trapping, of DNA prism, etc.
However, all the above methods have the disadvantage of requiring a relatively long separation time, sometimes the use of very low, unconventional saline concentrations, and more generally of being complex to carry out and having insufficient resolution over a broad molecule size range.
There is therefore a need to develop a method for separating molecules or other objects without a separating matrix, which method requires a shorter separation time and is more simple and flexible to carry out than the methods of the prior art.